Post-translational modifications, such as ADP-ribosylation, are critical to the control of regulatory and metabolic pathways. The extent of ADP-ribosylation of arginine residues in proteins is governed by NAD: arginine ADP-ribosyltransferases, which covalently link the ADP-ribose moiety of NAD to proteins, and ADP-ribosylarginine hydrolases (ADPRH), which cleave the ADP-ribose-arginine bond, releasing ADP-ribose and regenerating free arginine. (a) Characterization of the ADP-ribosylarginine hydrolase gene: To elucidate regulation of the ADP-ribosylarginine hydrolase gene, the full length mouse cDNA was cloned, intron-exon boundaries were determined, and the promoter region was identified and characterized. The hydrolase gene spans 7.8 kb, with 4 exons. The promoter region is GC rich, consistent with the finding of multiple transcription initiation sites. Following transient transfection of rat PC12 pheochromocytoma cells, mouse NB41A3 glioblastoma cells, and mouse NIH3T3 cells with wild-type and mutated promoters linked to a luciferase construct, stimulatory and inhibitory regions between -101 to -119 and -142 to -159, respectively, were identified. Mutational analysis and electrophoretic mobility shift assays (EMSA) defined a novel critical regulatory motif, AGCACC, involved in enhancing luciferase expression. The inhibitory element was localized to an 18-bp region; EMSA identified possible binding factors in nuclear extracts from each of the cell lines. Thus, the ADPRH gene contains a GC-rich promoter and inhibitory and stimulatory elements, which are utilized in diverse cell lines. (b) Characterization of the active site residues in ADP-ribosylarginine hydrolase: ADP-ribosylarginine hydrolases from mammalian tissues and\italicize {Rhodospirilium rubrum} exhibited regions of similarity in deduced amino acid sequences. It was postulated that amino acids in these areas could represent consensus domains critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a GST-fusion protein in\italicize {E. coli} and purified by glutathione-Sepharose affinity chromatography. Replacement of Asp60 or Asp61 with Ala, Gln, or Asn by site-specific mutagenesis significantly reduced enzyme activity, whereas mutants with a Glu replacement retained activity; the catalytically inactive mutants appeared to retain conformation since they bound ADP-ribose with affinity similar to that of the wild type. Replacing His65, Arg139, Asp285, which are also located in conserved regions, with alanine did not change specific activity. These data clearly show that Asp60 an Asp61 in rat ADP-ribosylarginine hydrolase are required for activity.